This episode examines a study that identifies recurrent neoantigens produced by SRSF2 and ZRSR2 splicing factor mutations in myeloid leukemias, isolates cognate TCRs, and demonstrates antigen-specific TCR-T cell activity in vitro and in vivo.
0:00Welcome to Base by Base, the papercast that brings Genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. So glad to be here. You know, um, when we look at the landscape of modern medicine, there are certain areas where the clinical statistics just completely stop you in your tracks.
0:19absolutely. And I want to talk directly to you, the listener, about a really stark reality in oncology today. We're talking about acute myloid leukemia or AML. And high risk mileid is plastic syndromes, you know, MDS.
0:34Right. These are just incredibly aggressive, devastating blood cancer. They really are. I mean, for literally half a century, the five-year survival rate for AML has stubbornly sat below 20%. Which is incredibly frustrating.
0:47Exactly. Because we've seen modern miracles in other hematological malignancies. Like we have car RT cell therapy, where we engineer the immune system to actively hunt down tumors. Right, but those modern miracles have largely failed in myeloid leukemias.
1:00Yeah, and the reason comes down to a biological bottleneck, right? Friendly fire. Exactly. The antigens or the molecular flags that sit on the surface of these leukemia cells are the exact same flags that sit on your healthy blood stem cells.
1:13So if you program the immune system to attack those flags. You eradicate the cancer, but you completely wipe out the patient's healthy bone marrow in the process. Yeah, it's a fundamental targeting problem.
1:25But what if the cancer cells made a predictable, systemic typo in their own instruction manuals that placed a unique neon flag right on their surface. How could this change our entire approach to eradicating leukemia?
1:38Today, we celebrate the work of Memorial Sloan Kettering Cancer Center and the Fred Hutchinson Cancer Center, who have advanced our understanding of targeted immunotherapies from Miled leukemias. And to really understand the mechanism driving these specific leukemias.
1:51We have to look deep inside the cell's RNA processing machinery. We do, because 50 to 70% of MBS patients and a massive portion of AML patients share mutations in a very specific set of genes. We're looking at RNA splicing factors, right?
2:06Specifically mutations in genes named SRSF2 and ZRSR2. Right. And, you know, the RNA splicing process is incredibly highly regulated. Has to be. I mean, the cell doesn't just indiscriminately paste Exxons together.
2:19Exactly. The spice of some is this massive dynamic complex. It relies on these splicing factors to recognize specific sequence motifs on the pre-MRNA, basically to determine exactly where to cut. Okay, so when SRSF2 or ZRSR2 are mutated, the physical structure of that recognition complex changes.
2:39Yeah, it does. Wait, mutations usually create random, unpredictable chaos. If every cancer is unique, how does random chaos give us a reliable target we can use for multiple patients? Well, the underlying biology here defies that instinct.
2:53It's really fascinating. These specific splicing factor mutations are neomorphic. Neomorphic. Yeah, they don't just represent a simple loss of function where this play system just falls apart and stops working.
3:02Oh, I see. So it doesn't just break. Right. Instead, the mutation offers the binding affinity of the splicing factor for specific RNA sequences. Okay, so it changes what it's looking for. Exactly. For example, a mutation at a specific amino acid residue in SRSF2 changes the complex, so it preferentially binds to a slightly different RNA motif.
3:22And because it's binding to a different motif, it forces the splice system to make highly specific, stereotyped, misplacing errors. You've got it. And the crucial point for you to understand is that because the genetic mutation is identical from patient to patient, driven by the same selective pressure in the cancers evolution.
3:40Right, exactly. The resulting missplicing event is also identical across different patients. Wow. So it acts less like a hammer smatching of printing press and more like, I don't know, a software glitch that forces the press to systematically misspell the exact same word on the exact same page over and over again.
3:57That is a perfect analogy. And because the transcript typo is identical. The resulting translated protein is also identical across the patient population. Right. And when the cell processes that mutated protein and presents fragments of it on the cell surface via the HLA system, you get what's called a public neo antigen.
4:14Yes. It is an off the shelf target present in anyone whose leukemia carries that specific neomorphic splicing mutation. Which is huge, right? Because the implications of a public neo-antigen are just staggering for drug development.
4:28Oh, absolutely staggering. We are no longer talking about sequencing an individual patient's tumor to manufacture of bespoke custom therapy just for them. Which is incredibly expensive and time consuming.
4:40Exactly. A public near antigen allows for the development of a single, highly refined, targeted therapy that can be administered to huge swaths of the patient population. But finding these sheer targets in the 1st place, I mean, that's like looking for a needle in a haystack.
4:55It really is. The researchers had to execute a massive computational dragnet before they ever touched a test tube. Right, because we're talking about massive RNA sequencing cohorts. Huge ones. They utilize data from the Can Genome Atlas, the Lusigine cohort, and the beat AML cohort.
5:11We are looking at 100s of patient transcriptums here. Okay, so they analyze the RNA of leukemias, harboring these specific splicing mutations against leukemias with wild type or normal splazing factors.
5:24Right. And crucially, they also mapped this against transcriptomic data from 14 different normal human tissues. Oh, so they wanted to be absolutely sure. Yeah, they were hunting for misspliced RNA isoforms that were highly expressed in the cancer, but rigorously completely absent in healthy biology.
5:42But, you know, a computational model predicting an RNA transcript is basically just a hypothesis. Very true. I mean, the algorithm says these neon flags should theoretically exist, but biological reality is notoriously messy.
5:55Oh, definitely. Getting from a misplaced RNA transcript to a physical peptide displayed on the surface of a leukemia cell requires surviving an intense cellular obstacle course. Right, because not every RNA is translated and not every protein is cleaved properly by the proteisone to actually fit into an HLA groove.
6:12Exactly. So how did they bridge the gap from in silico prediction to proving these flags physically exists? How did they do that? Well, the transition required advanced predictive algorithms, specifically one called NetMHCPan 4.0.
6:26Okay, and what does that do? It predicts the binding affinity of peptide sequences to specific HLA alleges. The cell uses HLA molecules to display intracellular peptides to the immune system. Got it. And for this study, they focus intensely on HLA 802.01, right?
6:44Yes, which is a highly prevalent allele globally. The algorithm scoured the translated sequences of those misplaced transcripts to identify specific peptide fragments. Fragments that had the precise biochemical properties, like the right anchor residues, to bind stably within the groove of that specific HLA molecule.
7:04You've got it. That's exactly it. But even after predicting the exact peptide sequence that fits that groove, finding the physical T cells that naturally recognize that complex in a patient's blood is incredibly difficult.
7:15Oh, it's like finding a specific grain of sand on a beach. We are talking about isolating ultra rare T cells from a massive background of circulating immune cells. Right, and using standard monumeric peptide HLA complexes just wouldn't work, would it?
7:27No, because a single T cell receptor binding to a single HLA molecule has incredibly low affinity. The connection is too weak. So it would just detach during the physical wash steps of flow cytometry. Exactly.
7:40So they had to use dextrimers. Which is kind of like fishing with highly specific magnetic lures, right? to amplify the binding strength through avidity. That's a great way to put it. The Dextremer technology is essential here.
7:51A dextrimer is a sophisticated molecular scaffold, usually with a dextrin backbone, and it's loaded with multiple copies, often up to 20, of the specific peptide HLA complex. Oh, wow. Up to 20. Yeah. So while the affinity of a single T cell receptor for a single peptide HLA might be low, the Dextrimer allows for multivalent binding.
8:13Meaning multiple T cell receptors on the surface of a single T cell engaged simultaneously with multiple complexes on the Dextrimer. Right. This creates massive avidity. The cumulative binding strength just locks the T cell onto the Dextrimer.
8:26That's brilliant. And they also attach DNA barcodes and fluorophores to these scaffolds, right? They do, which allows them to use fluorescence activated cell sorting to physically pull these extremely rare neoantigen reactive T cells out of 1000000s of background cells.
8:40And then sequence them to identify their specific T cell receptors. Okay, so catching these cells is one thing. But what did they actually catch with these high tech lures? Well, the yield is fascinating.
8:50They started with 1000s of potential mispllicing events. Thousands, okay. But after filtering for tumor specificity, HLA binding prediction and physical TCL capture. They validated 56 highly specific candidates generated by the SRSF 2 mutation.
9:07Wow, 56 targets. And what about ZRSR 2? They validated 19 candidates for ZRSR2. That's a solid list of targets. But out of all of those, they heavily focused on a neoantigen derived from a gene called CLK3, right?
9:21Yes, CLK3 was the star player. And the biological mechanism that generates the specific target relies on an incredibly ironic self-sabotage by the cancer cell's own quality control system. Oh, I love this part.
9:34Tell us how that works. So the biogenesis of the CLK3 neoantigen is a textbook example of cellular biology being leveraged against the tumor. The SRSF2 mutation alters the splicing machinery, so that it systematically skips Exxon 4 in the CLK3 Messenger RNA.
9:51And this skipping event introduces a frame shift, which subsequently leads to a premature termination code on, a stop sign where there shouldn't be one. Exactly. Now, the cell has a robust internal proofreading mechanism to prevent truncated, potentially toxic proteins from accumulating.
10:05Right, because during the pioneer round of translation, the ribosome hits this premature stop code on well upstream of where it should be. Right. The abnormal spatial arrangement triggers a highly conserved pathway called nonsense mediated decay or MMD.
10:20So the cell essentially realizes it made a mistake, recognizes the RNA transcript is corrupted, and marks the resulting truncated protein for immediate destruction. Yes. The proteisum, which acts as the cell's molecular wood chipper.
10:33shreds the faulty seal K3 protein into tiny peptide fragments. But those fragments aren't just expelled as waste. Nope. They are picked up by the TAP transporter system. Shuttle directly into the endoplasmic reticulum, loaded onto newly synthesized HLA molecules, and push straight to the surface of the cell.
10:51That is wild. The tumor's frantic attempt to clean up its own mispllicing mess is exactly what manufactures the neon flag and waves it on the cell surface. It's beautiful, really. And the efficiency of that processing pathway is what makes the CLK3 target so dense on the cell surface.
11:09Okay, so they have the target. What about the TCL receptors they isolated? When they sequence the TCL receptors capable of binding to this CLK3 fragment? They discovered receptors with staggering potency.
11:21How potent are we talking? Well, we measure molecular binding interactions using the dissociation constant, where a lower number indicates a tighter, more stable interaction. Most natural T cell receptors bind to their targets with micromolar or nanomolar affinity.
11:35The TCRs they isolated for the CLK3 neoanigen exhibited picomolar affinity. Pico molar affinity. That is orders of magnitude stronger than typical immune recognition. It really is. It means the off rate is incredibly slow.
11:48Once that engineered T cell receptor encounters the CLK3 peptide HLA complex, it chemically locks on and refuses to let go. Wow. So it requires an infinitesimally small number of targets on the leukemia cell surface to reach the threshold for immune activation.
12:04Exactly. And they demonstrated the lethal efficiency of this type binding in xenograph mouse models. Right. So they took human tea cells, engineered them to express these pico molar affinity TCRs, and introduced them into mice ingrafted with the human SRSF 2 mutant leukemia.
12:21And the data was spectacular. These T cells engaged in a highly targeted assassination of the cancer, drastically reducing the tumor burden without exhibiting any off target toxicity against healthy tissue.
12:32Which proves the target is completely cancer specific. But wait, this brings up a massive biological paradox. It does, yes. Let's look at the actual leukemia patients walking around right now. The sequencing proves they have the SRSF 2 mutation.
12:44The NMD pathway proves their cancer cells are actively churning out and displaying these CLK3 and RHOT2 neo-andigens. And the flow sitometry proves their bodies are successfully generating T cells, with receptors perfectly built to recognize these flags.
13:00So patients already have these neon flags on their cancer cells, and the body can clearly make T cells to recognize them. Why are the patients still sick? Why isn't the immune system fighting back naturally and clearing the leukemia?
13:14The resolution to that paradox provides one of the most clinically profound insights of the entire study. The researchers didn't just count the T cells in the patient blood. They performed single cell RNA sequencing.
13:26coupled with paired TCR sequencing, right? Exactly. This technology allows you to look at the global gene expression profile of the exact individual T cell that is reacting to the neo-antigen. And what did that transcript toomic data reveal?
13:39It revealed that the patient's immune system does see the flag. The T cells are physically present in the tumor micro environment. However, they are fundamentally broken. They are trapped in a state of terminal exhaustion.
13:51Oh, wow. Exhaustion. And that concept goes far beyond them simply being like suppressed, doesn't it? Oh, absolutely. The leukemia is not a fleeting viral infection. It is a chronic, relentless presence.
14:05These specific T cells are subjected to continuous, unabated antigen stimulation. Right, and over time, that constant activation fundamentally alters the epigenetic and transcriptional landscape of the T cell.
14:17Yes. The single cell data highlighted a profound impairment in the NFKB signaling pathway within these specific neoantigen reactive cells. And NFKB is a master transcription factor. When a healthy T cell receptor binds its target, NFKB translocates to the nucleus to trigger the massive cytokine cascade.
14:37Exactly. The release of IL2 interfere on gamma and the cytotoxic grandzymes needed to punch holes in the cancer cell. But in the leukemia patients that critical circuitry is burned out. Ah, so the chronic exposure upregulates inhibitory receptors and blocks the NFKB signaling bottleneck.
14:54Precis. The T cells are bumping directly into the leuchemic blasts. The receptors are engaging the misplaced neoantigens, but the intracellular alarm bell is completely disconnected. They are physically incapable of mounting a cytotoxic response.
15:05Yes. The target is valid, and the recognition is valid. But the biological execution fails because the local microenvironment has driven the attacking cells into terminal exhaustion. But there is incredible proof that the targets actually do work, right?
15:21The researchers found definitive clinical proof that if you bypass that exhaustion, the target works flawlessly. They really did. They analyzed a cohort of patients who had undergone curative, allogenic stem cell transplants.
15:34Right. So in this procedure, the patient's diseased bone marrow, along with their exhausted immune system is chemically or radiologically wiped out. And then they are engrafted with healthy donor stem cells.
15:45When the researchers profiled the blood of these patients post-transplant, they observed a spectacular immunological event. The healthy donor T cells flooded the patient's system. And because these donor cells were completely fresh, their NFKB pathways were pristine and highly responsive.
16:01Exactly. The transcriptomic profiles of the donor T cells reacting to targets like the RHOT2 misspliced neoantigen were night and day compared to the pretransplant patient cells. The donor cells exhibited hyperactivated, highly cytotoxic gene signatures.
16:17They did. They successfully recognized the misspliced RNA flags, expanded massively in number, and exerted fierce selective pressure against the leukemia. So this post-transplant data unequivocally proves that the RNA misplacing neo antigens are highly immunogenic natural targets.
16:35Yes, the failure in the native state is purely a function of T cell exhaustion, not a flaw in the antigen itself. The clinical gravity of this finding is just immense. I mean, this is a literal, validated blueprint for off the shelf, genetically redirected TCRT cell therapies for blood cancers that desperately need them.
16:54It really is. We can finally bypass the friendly fire problem that has plagued Kara T in AML. The vision here is to harvest a patient's tea cells, extract them from that suppressive microenvironment, genetically engineer them to express these highly specific pecomolar affinity TCRs.
17:09Right, against CLK 3 or other misplliced targets. Culturally rejuvenate them and infuse an army of highly potent, unexhausted cells back into the patient. And they will exclusively hunt the leukemic blasts bearing the neomorphic mutation, leaving the healthy, unmutated, hematopoietic stem cells completely alone.
17:27It's an incredible blueprint. But translating this structural biology and early mouse data into human clinical reality requires balancing the excitement with scientific rigor. Absolutely. We have to look at the limitations.
17:39The primary constraint of this foundational study is its heavy focus on one immune type, HLA, A02.01. Right, because the HLA system is the most polymorphic gene cluster in the human genome, while A02.01 is prevalent in certain demographics, relying solely on it excludes a massive portion of the global patient population.
18:01Yes. Future therapeutic development must broaden the in silico predictions and physical validations to discover misplaced targets for other HLA types globally to ensure equitable access. The display window has to match the patients' specific genetics.
18:14And also, while the early mouse models are incredibly promising, they rely on immunodeficient mice to accept the human leukemia graph. Which means these models cannot fully replicate the complex, immunosuppressive human tumor microenvironment.
18:27So to truly optimize how long these engineered T cells can survive in the body, and whether they can avoid falling back into exhaustion, the field needs advanced immunocompetent humanized models. Yes, ultimately leading into carefully designed phase I clinical trials.
18:43Right. Now, looking at the trajectory of this biology, I want to build on the text with a forward looking thought. Splicing mutations, particularly in SRSF 2, frequently occur as early initiating events in pre-leukemic conditions.
18:56Yes, like clonal hematopoises. Exactly. The genetic typo, and theoretically, the resulting misspliced RNA flags, are present in the marrow long before the full-blown acute cancer develops. That's great point.
19:09So if these shared neo antigens are truly highly immunogenic. Could we theoretically use these neo-antigens as a vaccine to train the immune system before the leukemia fully develops. Targeting clonal hematopoiasis with a preventative neoantigen vaccine is a highly sophisticated direction for future research.
19:25makes sense, right? Immunize patients utilizing their still healthy immune system to intercept and eradicate the mutant clones. Because these neomorphic mutations are shared and occur early, intercepting the premalignant state before widespread immune exhaustion sets in, represents a massive therapeutic window.
19:43It's just brilliant. So bringing this deep dive to a cohesive close for you, the listener. Mutations in RNA splicing factors create highly predictable shared neo antigens on the surface of leukemia cells.
19:56By engineering healthy T cells to recognize these specific misspliced targets, we can selectively eradicate cancer cells while entirely sparing healthy tissue. What does this mean for the future of treating other cancers driven by seemingly undruggable mutations?
20:10It's going to be incredible to watch unfold. This episode was based on an open access article under the CCBY 4.0 license. You can find a direct link to the paper and the license in our episode description.
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